Methods: :
Four stereoisomeric dipeptide prodrugs L-Valine-D-Valine-Gancyclovir(LDGCV), D-Valine-L-Valine-Gancyclovir (DLGCV), L-Valine-L-Valine-Gancyclovir(LLGCV), D-Valine-D-Valine- Gancyclovir (DDGCV) and two aminoacid prodrugs D-Val-Gancyclovir (DGCV), and L-Val-Gancyclovir(LGCV) were synthesized and characterized using mass and NMRspectrometry. MDCK and SIRC cell lines were used as model celllines to study the interaction of these prodrugs with PeptideTransporter (PEPT). The aqueous stability studies of these prodrugswere performed in Dulbecco’s phosphate buffered Saline(DPBS) at different pH (6.5, 7.4 and 8.5) at 340C for 10 days.Metabolism of these prodrugs were carried out in ocular tissuehomogenates. Cytotoxicity of the prodrugs was analyzed usingCtoTox-ONETM Homogenous Membrane Integrity Assay kit from Promega.Corneal transport of these prodrugs was carried out using aside-by-side diffusion apparatus. All the samples were analyzedusing HPLC and LC-MS/MS.

Results: :
No detectable degradation was observed for LDGCV, DGCV at pH6.5. However, the degradation rate was found to be higher atalkaline pH. The stability in increasing order for the prodrugswas found to be DDGCV>LDGCV>DLGCV>DGCV>LLGCV>LGCV.The interaction of these prodrugs with PEPT was confirmed bytheir significant inhibition of [3H] Glysar uptake in vitro.All prodrugs other than DDGCV and DGCV were found to interactwith PEPT1. Cytotoxicity of the prodrugs was significantly lessthan the marketed drug Trifluorothymidine. Corneal transportsample concentrations were not detectable by HPLC detectionlimit. Extraction and analytical method were optimized in LC-MS/MSto analyze these samples.

Conclusions: :
Our results indicate that stereoisomeric dipeptide monoesterprodrugs of GCV can not only enhance the aqueous stability butalso retain their affinity towards Peptide transporter as wellas improving the enzymatic stability. Hence these can be a bettersubstitute to conventional peptide prodrugs of GCV in treatmentof Herpes Simplex virus infections.